The present invention relates to fluid handling systems and, particularly, to methods and apparatus for depositing biological materials in a pattern of array features on a surface of a solid support.
Chemical and biological research, development, and manufacturing, in fields such as combinatorial chemistry, genomics, and proteomics, often requires the simultaneous handling of small quantities of many different fluids including gases and liquids. Gases can often be handled easily using tubing and manifolds, but liquid handling is often difficult.
Liquid samples are often handled and stored in microtiter plates. Microtiter plates are rectangular trays made of glass or plastic. They contain many small liquid reservoirs adjacent to one another for reacting and storing liquids in typical arrays sizes of 96 in an 8xc3x9712 array of 400 microliter (xcexcl) wells on 9 millimeter (mm) spacing, 384 in a 16xc3x9724 array of 100 xcexcl wells on 4.5 mm spacing, or 1536 in a 32xc3x9748 array of 10 xcexcl wells on 2.25 mm spacing. Transferring the many liquid samples from microtiter plates to other formats such as microarrays presents many challenges.
Microarrays of binding agents have become an increasingly important tool in the biotechnology industry and related fields. Such arrays, in which such binding agents as oligonucleotides or peptides are deposited onto a solid support surface in the form of an array or pattern, can be useful in a variety of applications, including gene expression analysis, drug screening, nucleic acid sequencing, mutation analysis, and the like.
Such arrays may be prepared in any of a variety of different ways, many of which rely on transferring liquids from an array of liquid samples in one or more microtiter plates to the substrate on which the microarray is formed. For example, DNA arrays may be prepared manually by spotting DNA onto the surface of a substrate with a micropipette. Or, a dot-blot approach or a slot-blot approach may be employed in which a vacuum manifold transfers aqueous DNA samples from a plurality of reservoirs to a substrate surface. Or, an array of pins can be dipped into an array of fluid samples and then contacted with the substrate surface to produce the array of sample materials. Or, an array of capillaries can be used to produce biopolymeric arrays.
In an alternative approach, arrays of biopolymeric agents are constructed in discrete regions on the surface of the substrate.
There is a continued interest in developing methods and devices for making arrays of biomolecules, in which the apparatus is less complicated and more automated and the methods reduce waste of biological material that may be in limited supply, and which result in efficient and reproducible rapid production of more versatile and reliable arrays.
Inkjet printing devices have been modified and used to dispense biochemical agents such as proteins and nucleic acids but have not been able to achieve a spatial density of liquid samples at the inkjet head comparable to the spatial density of ink ejection orifices on the head itself. Thus, even though the orifices on an inkjet head may be spaced less than 100 micrometers (xcexcm) apart from one another, the spacing of different liquid samples feeding such a head has not been reduced to less than millimeters (mm).
Recent art taught in international patent application WO9955461(A1) discloses a redrawn capillary imaging reservoir which may be used for pin-printing of liquid samples onto a microarray and which also may be used in transferring liquid samples between microtiter plates of different well density. However, the art taught in that patent application results in relatively large volumes of liquid required to fill the capillary system taught therein. Since the liquid samples used for microarray fabrication are often scarce and expensive, the requirement for large filling volumes can create problems is practicing that art.
Thus there still exists a need for a droplet deposition system which is fed from hundred of different reservoirs, which can deposit hundreds of different fluids in the form of drop-on-demand droplets onto substrates for purposes such as microarray fabrication, and
The present invention provides a fluid handling system, and a method of manufacture therefor, having a flexible manifold including two layers of flexible material laminated together. The flexible manifold has provided therein a capillary, a capillary inlet hole fluidically connected to the capillary, and a capillary outlet hole fluidically connected to the capillary. A peripheral rim is attached to the flexible manifold and exerts tensile stress on the flexible manifold to provide dimensional stability to the flexible manifold.
The present invention further provides a fluid handling system allowing hundreds of different liquids to supply an edge-fed, drop-on-demand, face-shooting, thermally-actuated xe2x80x9cdeposition chipxe2x80x9d from a supply region comprising hundreds of reservoirs wherein each reservoir has a volume on the order of microliters. The deposition chip is a modified silicon inkjet chip placed upon a stretched microfluidic structure called a xe2x80x9cformat compression manifoldxe2x80x9d (FCM). The FCM contains hundreds of capillaries, orifices, and feedthrough holes which allow fluidic transfer of hundreds of different liquid samples from the format spacing of several millimeters, as typically used in microtiter plates, to a format spacing of tens of micrometers, as typically used in inkjet orifices originally developed for printing with ink on paper. The deposition chip then spits each of the hundreds of different liquids as individual droplets with a volume on the order of picoliters, in a drop-on-demand mode, onto substrates such as glass plates. Such plates can comprise microarrays such as DNA microarrays or protein microarrays which can contain thousands of different spots of thousands of different biological samples. The FCM is provided with dimensional stability by being stretched like a drumhead on a rigid frame called the xe2x80x9crim.xe2x80x9d
An additional plate known as a microtiter manifold (MTM) is adhesively bonded over inlet holes at the ends of the FCM capillaries distal from the deposition chip. The MTM contains hundreds of liquid reservoirs laterally spaced on centers of several millimeters, with each reservoir being in fluid communication with one capillary of the FCM. In variations of this embodiment, multiple MTMs may be bonded onto the FCM and multiple deposition chips may be bonded onto the FCM.
In a second embodiment of the present invention the MTM and the rim form one contiguous body which is adhesively bonded to the openings of the FCM capillaries distal from the deposition chip, which contiguous body contains the hundreds of liquid reservoirs, and which acts to keep the FCM stretched for dimensional stability. In a third embodiment of the present invention a separate microtiter plate is mechanically clamped in place to align it to inlet holes of the FCM capillaries distal from the deposition chip, such that each reservoir of the microtiter plate is in fluid communication with one capillary of the FCM. Gasketing between the microtiter plate and the FCM prevents cross-leakage between the reservoirs of the microtiter plate. In variations of this embodiment, multiple microtiter plates may be clamped onto the FCM, and multiple deposition chips may be bonded onto the FCM.
An advantage of the present invention is that it takes separate fluid samples, which are initially loaded into reservoirs, which are laterally spaced several millimeters apart, and decreases the spacing between the separate samples to tens of micrometers before the samples are spit onto substrates where the desired spacing between samples is also tens of micrometers. This decrease in spacing is called xe2x80x9cformat compressionxe2x80x9d, and is expressed in a figure of merit called the format compression ratio (FCR). For example, in one embodiment of the invention the lateral spacing between samples when loaded into reservoirs is 2.25 mm, while the lateral spacing between samples at the deposition chip is reduced to 84.7 um. The FCR is thus 2.25 mm=2250 um divided by 84.7 um, which is equal to 26.6. When the rows of the firing chambers along opposite lateral edges of the deposition chip are offset from one another, either by chip design or by slightly rotating the deposition chip with respect to the substrate on which it is to deposit spots, the FCR can be doubled to 53. This permits the correspondence of one reservoir to one firing chamber at spacings, which have not been achievable with prior technology.
Another advantage of the invention is minimal priming volume. The multiple incoming liquid samples can be expensive, and when large volumes of such samples are required in order to prime a deposition system before its use, then deposition costs rise correspondingly. The present invention minimizes the priming volume and so keeps deposition costs low. Typical priming volumes for the present invention are in the range of 100 nanoliters (nl) to 400 nl.
Further, when incoming samples arrive at the manufacturing site in industry-standard load formats such as those of a 96-well microtiter plate, a 384-well microtiter plate, or a 1536- well microtiter plate, cost savings arise when the handling expense of transferring the incoming samples to the fluid handling system is minimized. When an incoming microtiter plate can be loaded directly into the fluid handling system through a specialized filter plate without first removing the many incoming samples from the microtiter plate, the load format compatibility is maximized as in the present invention.
Another advantage of the invention is dimensional stability. Despite being constructed using a flexible polymer which tends to undergo shrinkage during fabrication, the present invention achieves dimensional stability by stretching the rigid polymer, during fabrication, on a rigid frame. The polymer stretched on the frame attempts to shrink, but its shrinkage is prevented by the rigid frame, and the result is a structure in which the polymer is stretched like a drumhead, so that in addition to dimensional stability in the plane of the polymer, the tautness of the stretched polymer provides stability perpendicular to the plane of the polymer. Dimensional stability in all three physical directions allows both precision in placing the deposition chip on the FCM in relation to ejection orifices machined into the FCM and precision in deposition of droplets onto substrates.
Further, the peripheral rim in the present invention, like the rim of a drumhead, serves to keep the flexible format compression manifold flat and taut. However, unlike a drumhead, the FCM need not be flat and taut everywhere, and instead may have external hanging flaps, internal hanging flaps, and unrestrained internal portions. The rim around the FCM need not extend completely around the FCM periphery, nor need it extend completely around any MTM on the FCM. The rim needs only to extend far enough around an area where tautness is desired to keep that area taut. Cutout regions and unrestrained flaps can be created in the FCM either before or after the layers of the FCM are laminated to the rim. The rim may be split, either before or after lamination, into more than one portion.
The above and additional advantages of the present invention will become apparent to those skilled in the art from a reading of the following detailed description when taken in conjunction with the accompanying drawings.